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Abstract:

Herein is reported a fusion polypeptide comprising i) at least one
binding site, e.g. which comprises an antibody heavy chain variable
domain and an antibody light chain variable domain, and which binds to an
internalizing cell surface receptor, and ii) at least one
pharmaceutically active compound, whereby the EC50-value of the
binding pair that binds to an internalizing cell surface receptor
determined at pH 5.5 is higher than the EC50-value of the same
binding pair determined at pH 7.4 and its use for delivering a
pharmaceutically active compound across the blood-brain-barrier.

Claims:

1. A method of delivering a pharmaceutically active compound across the
blood-brain-barrier in an individual comprising administering to the
individual an effective amount of a fusion polypeptide comprising at
least one binding pair, which comprises an antibody heavy chain variable
domain and an antibody light chain variable domain, and which binds to an
internalizing cell surface receptor, and at least one pharmaceutically
active compound, whereby the ratio of the EC50-value of the binding
pair that binds to an internalizing cell surface receptor determined at
pH 5.5 and the EC50-value of the same binding pair to the same
receptor determined at pH 7.4 is 10 or more, such that the fusion
polypeptide delivers the pharmaceutically active compound across the
blood-brain-barrier.

2. (canceled)

3. A method of transcytosing epithelial cells of a subject comprising
administering to the subject a fusion polypeptide comprising at least one
binding pair, which comprises an antibody heavy chain variable domain and
an antibody light chain variable domain, and which binds to an
internalizing cell surface receptor, and at least one pharmaceutically
active compound, whereby the ratio of the EC50-value of the binding
pair that binds to an internalizing cell surface receptor determined at
pH 5.5 and the EC50-value of the same binding pair to the same
receptor determined at pH 7.4 is 10 or more.

4. A fusion polypeptide comprising at least one binding pair, which
comprises an antibody heavy chain variable domain and an antibody light
chain variable domain, and which binds to an internalizing cell surface
receptor, and at least one pharmaceutically active compound, whereby the
ratio of the EC50-value of the binding pair that binds to an
internalizing cell surface receptor determined at pH 5.5 and the
EC50-value of the same binding pair to the same receptor determined
at pH 7.4 is 10 or more.

5. A method of increasing transport of at least one pharmaceutically
active compound across the blood-brain-barrier in an individual relative
to the transport across the blood-brain-barrier of an unconjugated form
of the one or more pharmaceutically active compound, comprising
administering to the individual an effective amount of a fusion
polypeptide comprising at least one binding pair, which comprises an
antibody heavy chain variable domain and an antibody light chain variable
domain, and which binds to an internalizing cell surface receptor, and
the at least one pharmaceutically active compound, whereby the ratio of
the EC50-value of the binding pair that binds to an internalizing
cell surface receptor determined at pH 5.5 and the EC50-value of the
same binding pair to the same receptor determined at pH 7.4 is 10 or
more, such that the fusion polypeptide transports the pharmaceutically
active compound across the blood-brain-barrier.

6. A method of selecting a binding pair for use in efficient
blood-brain-barrier transport of one or more pharmaceutically active
compounds comprising measuring a ratio of the EC50-values for
binding of one or more binding pairs to an internalizing cell surface
receptor at pH 5.5 and pH 7.4, and selecting one or more binding pairs
wherein the ratio is 10 or more.

7. (canceled)

8. The method of claim 20, characterized in that the CNS-related disease
is selected from (i) neurodegenerative diseases or disorders selected
from Parkinson's disease, Alzheimer's disease, and Huntington's disease;
(ii) psychiatric diseases selected from depression, anxiety disorders,
and schizophrenia; (iii) neuroinflammatory and other neurological
disorders selected from multiple sclerosis, Amyotrophic Lateral
Sclerosis, autism, and pain; (iv) tumors of the CNS, and (v) viral and
bacterial infections of the CNS.

9. The method, fusion polypeptide or pharmaceutical formulation of any
one of claims 1, 3-6 and 19 wherein the ratio is selected from about 15
and 15 or more, or wherein the EC50-value determined at pH 5.5 is
1000 ng/ml or more.

10. (canceled)

11. (canceled)

12. The method, fusion polypeptide or pharmaceutical formulation of any
one of claims 1, 3-6 and 19, wherein the binding pair is selected from an
Fv, a Fab, a Fab', a Fab'-SH, a F(ab')2, diabody, a linear antibody,
a single-chain antibody, a multispecific antibody, a full length heavy
chain, a full length light chain, a complete antibody, a bispecific
antibody, a trispecific antibody, a tetraspecific antibody, and a
hexaspecific antibody.

13. The method, fusion polypeptide or pharmaceutical formulation of any
one of claims 1, 3-6 and 19, wherein the pharmaceutically active compound
is attached to the at least one binding pair by a linker, or wherein the
pharmaceutically active compound is directly fused to the at least one
binding pair.

14. (canceled)

15. The method, fusion polypeptide or pharmaceutical formulation of any
one of claims 1, 3-6 and 19, wherein the internalizing cell surface
receptor is selected from a sialoglycoprotein receptor, an
alpha(2,3)sialoglycoprotein receptor, a diphtheria toxin receptor a
heparin-binding epidermal growth factor-like growth factor, a folate
receptor, a glutamate receptor, a glutathione receptor, an insulin
receptor, an insulin-like growth factor receptor, a leptin receptor, a
low-density lipoprotein receptor, an LDL-related protein 1 receptor, an
LRP2 receptor, an LRP4 receptor, an LRP5 receptor, an LRP6 receptor, an
LRP8 receptor, a mannose 6-phosphate receptor, a scavenger receptor
(class A or B, types I, II or III, or CD36 or CD163), a substance P
receptor, a thiamine transporter, a transferrin-1 receptor, a
transferrin-2 receptor, and a vitamin B12 receptor, or wherein the
internalizing cell surface receptor is a transferrin receptor.

16. (canceled)

17. The method, fusion polypeptide or pharmaceutical formulation of any
one of claims 1, 3-6 and 19, wherein the pharmaceutically active compound
is selected from a drug, a label, a cytotoxin, an enzyme, a growth
factor, a transcription factor, a radionuclide, a ligand, a liposome, a
nanoparticle, a viral particle, a cytokine, and an antibody or active
fragment thereof.

18. (canceled)

19. A pharmaceutical formulation comprising the fusion protein of claim
4, optionally with a pharmaceutically acceptable carrier or with at least
one additional therapeutic agent.

20. A method of treating a CNS-related disease using the pharmaceutical
formulation of claim 19.

Description:

RELATED APPLICATIONS

[0001] This application claims the benefit of European Patent Application
No. 11163200.6 filed on Apr. 20, 2011, which is incorporated by reference
herein in its entirety.

BACKGROUND

[0002] Herein is reported a fusion polypeptide comprising at least one
binding site and at least one pharmaceutically active compound, whereby
the EC50-value of the binding site that binds to an internalizing
cell surface receptor determined at pH 5.5 is higher than the
EC50-value of the same binding site determined at pH 7.4, and its
use for delivering a pharmaceutically active compound across the
blood-brain-barrier.

BACKGROUND OF THE INVENTION

[0003] Endothelial or epithelial cell layers interconnected by tight
junctions represent a major hurdle for the diffusion of large, polar
molecules, especially proteins, into the tissues behind these barriers.
While small molecules can be transported across these barriers by
specialized channel proteins, the transport mechanisms for proteins are
still incompletely understood, but the physiologically most important
mechanism is thought to be receptor-mediated transcytosis (RMT).

[0004] During RMT a protein ligand binds to a receptor expressed on the
luminal side of the barrier cells, which is then internalized by
endocytosis. Sorting of endosomal content is achieved in specialized
vesicular compartments and depends on signals encoded by the receptor
sequence, which mediate trafficking of the receptor into recycling,
degradation, or transcytosis pathways. One of the best known examples of
RMT is the transport of IgG across intestinal epithelial cells by the
neonatal Fc receptor in rodents.

[0005] Also for the blood-brain-barrier (BBB), which consists of tightly
sealed brain endothelial cells surrounded by pericytes and astrocytes,
several RMT pathways have been described, especially the receptors for
transferrin, insulin, or low-density lipoprotein. The ligands of these
receptors have been shown to harbor properties facilitating transcytosis,
one of these properties being pH-dependent binding to their receptors.
Insulin, for example, is released from its receptor upon acidification of
the endosomal content after internalization.

[0006] Researchers have attempted to exploit transcytosis of receptors for
the delivery of therapeutic molecules over the blood-brain-barrier by
coupling therapeutics to antibodies against these receptors. However,
none of these antibodies has been used in a marketed drug yet, probably
because of their insufficient transport potential. In fact, unequivocal
demonstration of functional therapeutic protein transcytosis over the
BBB, shown independently in more than one pharmacodynamic model, is still
missing.

[0008] In U.S. Pat. No. 6,030,613 the receptor specific transepithelial
transport of immunogens is reported. In WO 02/060919 are reported
molecules with extended half-lives, compositions and uses thereof. A
process for the preparation of transferrin receptor specific
antibody-neuropharmaceutical or diagnostic agent conjugates is reported
in WO 93/010819. In WO 2008/119096 transcytotic immunoglobulin are
reported. A human blood brain barrier model is reported in WO
2006/056879.

[0010] Herein is reported that a pH-dependent binding mode enables
antibodies directed against internalizing cell surface receptors,
especially transcytosis receptors, to efficiently cross a tight layer of
barrier cells, especially the blood-brain-barrier. It is shown that an
antibody, e.g. binding to the human transferrin receptor as an example of
an internalizing cell surface receptor, which has a low binding affinity
at pH 5.5 (higher EC50 value) as compared to its affinity at pH 7.4
(lower EC50 value), is transcytosed through blood-brain-barrier
endothelial cells, whereas a different antibody showing about equal
affinity (binding efficiency, and, thus, EC50 values) at both pH
values, is degraded inside the cells. Thus, the EC50-value of the
binding site that binds to an internalizing cell surface receptor
determined at pH 5.5 is higher (bigger) than the EC50-value of the
same binding site determined at pH 7.4. This allows the generation and
selection of antibodies against transcytosis receptors that are not
intracellularly degraded in endothelial or epithelial barrier cells due
to a modified sorting behavior caused by pH-dependent, reversible binding
to those receptors.

[0011] Herein is reported as one aspect a fusion polypeptide comprising
[0012] at least one binding site, which binds to an internalizing cell
surface receptor, and [0013] at least one effector moiety, [0014] whereby
the EC50-value of the binding site that binds to an internalizing
cell surface receptor determined at pH 5.5 is higher than the
EC50-value of the same binding site determined at pH 7.4.

[0015] In one embodiment the fusion polypeptide is characterized in that
the ratio of i) the EC50-value of the binding site that binds to an
internalizing cell surface receptor determined at pH 5.5 and ii) the
EC50-value of the same binding site to the same receptor determined
at pH 7.4 is at least 5. In one embodiment the ratio is 10 or more. In
one embodiment the ratio is 15 or more. In one embodiment the ratio is
about 15.

[0016] In one embodiment the EC50-value of the binding site that
binds to an internalizing cell surface receptor determined at pH 5.5 is
at least 5-times the EC50-value of the same binding site to the same
receptor determined at pH 7.4. In one embodiment the EC50-value
determined at pH 5.5 is at least 10-times the EC50-value determined
at pH 7.4. In one embodiment the EC50-value determined at pH 5.5 is
about 15-times the EC50-value determined at pH 7.4.

[0017] In one embodiment the effector moiety is a label, or a cytotoxin,
or an enzyme, or a growth factor, or a transcription factor, or a drug,
or a radionuclide, or a ligand, or an antibody, or antibody fragment, or
a liposome, or a nanoparticle, or a viral particle, or a cytokine

[0018] In one embodiment the effector moiety is a pharmaceutically active
compound. In one embodiment the pharmaceutically active compound is an
anti-Abeta antibody, or an anti-tau antibody, an anti-alpha synuclein
antibody, or an active fragment thereof.

[0019] In one embodiment the effector moiety is a pharmaceutically active
compound that is attached to the fusion polypeptide by a linker. In
another embodiment the effector moiety is a pharmaceutically active
compound that is directly fused to the fusion polypeptide.

[0020] In one embodiment the binding site that binds to an internalizing
cell surface receptor has an EC50-value determined at pH 5.5 of 100
ng/ml or more, or of 500 ng/ml or more, or of 1000 ng/ml or more.

[0021] In one embodiment the binding site that binds to an internalizing
cell surface receptor has an EC50-value determined at pH 7.4 of 100
ng/ml or less, or of 85 ng/ml or less, or of 70 ng/ml or less.

[0022] In one embodiment the binding site is a binding pair, which
comprises an antibody heavy chain variable domain and an antibody light
chain variable domain. In one embodiment the binding pair is selected
from an Fv, a Fab, a Fab', a Fab'-SH, a F(ab')2, a diabody, a linear
antibody, a single-chain antibody molecule, and a multispecific antibody
formed from antibody fragments, a full length heavy chain, a full length
light chain, a complete antibody, a bispecific antibody, a trispecific
antibody, a tetraspecific antibody, or a hexaspecific antibody. In one
embodiment the binding pair is a complete monoclonal antibody. In one
embodiment binding pair is at least a fragment of a complete antibody, a
member of the immunoglobulin superfamily, or a polypeptide with
immunoglobulin-like structure, that retains the binding specificity for
its antigen.

[0024] In one embodiment the internalizing cell surface receptor is
selected from a sialoglycoprotein receptor, an
alpha(2,3)sialoglycoprotein receptor, a diphtheria toxin receptor a
heparin-binding epidermal growth factor-like growth factor, a folate
receptor, a glutamate receptor, a glutathione receptor, an insulin
receptor, an insulin-like growth factor receptor, a leptin receptor, a
low-density lipoprotein receptor, an LDL-related protein 1 receptor, an
LRP2 receptor, an LRP4 receptor, an LRP5 receptor, an LRP6 receptor, an
LRP8 receptor, a mannose 6-phosphate receptor, a scavenger receptor
(class A or B, types I, II or III, or CD36 or CD163), a substance P
receptor, a thiamine transporter, a transferrin-1 receptor, a
transferrin-2 receptor, and a vitamin B12 receptor. In one embodiment the
internalizing cell surface receptor is a transferrin receptor.

[0025] Herein is reported as one aspect a nucleic acid encoding the fusion
polypeptide as reported herein.

[0026] Herein is reported as one aspect a host cell comprising the nucleic
acid as reported herein.

[0027] Herein is reported as one aspect a method of producing a fusion
polypeptide comprising culturing the host cell as reported herein so that
the fusion polypeptide is produced.

[0028] Herein is reported as one aspect a pharmaceutical formulation
comprising the fusion polypeptide as reported herein and optionally a
pharmaceutically acceptable carrier.

[0029] Herein is reported as one aspect the fusion polypeptide as reported
herein for use as a medicament.

[0030] Herein is reported as one aspect the fusion polypeptide as reported
herein for use in treating a CNS-related disease.

[0031] Herein is reported as one aspect the fusion polypeptide as reported
herein for use in delivering a pharmaceutically active compound across
the blood-brain-barrier.

[0032] Herein is reported as one aspect the use of the fusion polypeptide
as reported herein in the manufacture of a medicament.

[0033] In one embodiment the medicament is for treatment of a CNS-related
disease.

[0034] Herein is reported as one aspect a method of treating an individual
having a CNS-related disease comprising administering to the individual
an effective amount of the fusion polypeptide as reported herein.

[0035] Herein is reported as one aspect a method of delivering a
pharmaceutically active compound across the blood-brain-barrier in an
individual comprising administering to the individual an effective amount
of the fusion polypeptide as reported herein to deliver a
pharmaceutically active compound across the blood-brain-barrier.

[0036] Herein is reported as one aspect a method of delivering a
pharmaceutically active compound across the blood-brain-barrier to a
subject's brain, comprising administering a pharmaceutically active
compound fused to a binding pair, which comprises an antibody heavy chain
variable domain and an antibody light chain variable domain, and which
binds to an internalizing cell surface receptor, whereby the
EC50-value of the binding pair that binds to an internalizing cell
surface receptor determined at pH 5.5 is higher than the EC50-value
of the same binding pair determined at pH 7.4. In one embodiment the
fusion polypeptide is characterized in that the ratio of the
EC50-value of the binding site that binds to an internalizing cell
surface receptor determined at pH 5.5 and the EC50-value of the same
binding site to the same receptor determined at pH 7.4 is at least 5. In
one embodiment the ratio is 10 or more. In one the ratio is 15 or more.
In also an embodiment the ratio is about 15.

[0037] In one embodiment the EC50-value of the binding pair that
binds to an internalizing cell surface receptor determined at pH 5.5 is
at least 5-times the EC50-value of the same binding pair to the same
receptor determined at pH 7.4. In one embodiment the EC50-value
determined at pH 5.5 is at least 10-times the EC50-value determined
at pH 7.4. In one embodiment the EC50-value determined at pH 5.5 is
about 15-times the EC50-value determined at pH 7.4.

[0038] Herein is reported as one aspect the use of a fusion polypeptide as
reported herein for the delivery of a pharmaceutically active compound
across the blood-brain-barrier.

[0039] Herein is reported as one aspect a method of transcytosing
epithelial cells of a subject comprising administering to the subject a
fusion polypeptide as reported herein.

[0040] Herein is reported as one aspect a method of increasing transport
of at least one pharmaceutically active compound across the
blood-brain-barrier in an individual relative to the transport across the
blood-brain-barrier of an unconjugated form of the one or more
pharmaceutically active compound, comprising administering to the
individual an effective amount of a fusion polypeptide as reported herein
such that the fusion polypeptide transports the pharmaceutically active
compound across the blood-brain-barrier.

[0041] Herein is reported as one aspect a method for selecting an antibody
or a fusion polypeptide comprising at least one binding site wherein the
EC50-value of the antibody or fusion polyp eptide for binding to an
internalizing cell surface receptor determined at pH 5.5 is higher than
the EC50-value of the same antibody or the same fusion polypeptide
to the same receptor determined at pH 7.4.

[0042] In one embodiment the antibody or the fusion polypeptide is
characterized in that the ratio of the EC50-value of the binding
site that binds to an internalizing cell surface receptor determined at
pH 5.5 and the EC50-value of the same binding site to the same
receptor determined at pH 7.4 is at least 5. In one embodiment the ratio
is 10 or more. In one embodiment the ratio is 15 or more. In one
embodiment the ratio is about 15.

[0043] In one embodiment the EC50-value of the binding site that
binds to an internalizing cell surface receptor determined at pH 5.5 is
at least 5-times the EC50-value of the same binding site to the same
receptor determined at pH 7.4. In one embodiment the EC50-value
determined at pH 5.5 is at least 10-times the EC50-value determined
at pH 7.4. In one embodiment the EC50-value determined at pH 5.5 is
about 15-times the EC50-value determined at pH 7.4.

[0044] Herein is reported as one aspect a method for selecting a binding
pair for use in efficient blood-brain-barrier transport of one or more
pharmaceutically active compounds comprising measuring a ratio of the
EC50-values for binding of one or more binding pairs to an
internalizing cell surface receptor at pH 5.5 and pH 7.4, and selecting
one or more binding pairs wherein the ratio is 10 or more.

[0045] In one embodiment of all the aspects as reported herein the
CNS-related disease is selected from (i) neurodegenerative diseases or
disorders such as Parkinson's disease, Alzheimer's disease, or
Huntington's disease, or (ii) psychiatric diseases such as depression,
anxiety disorders, schizophrenia, or (iii) neuroinflammatory and other
neurological disorders such as multiple sclerosis, Amyotrophic Lateral
Sclerosis, autism, or pain, or (iv) tumors of the CNS, or (v) viral and
bacterial infections of the CNS.

DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention demonstrates that a pH-dependent binding mode
enables fusion polypeptides comprising at least one binding site and
antibodies directed against transcytosis receptors to efficiently cross a
tight layer of barrier cells. It is shown for example that an antibody
against the human transferrin receptor, which has a low binding affinity
at pH 5.5 as compared to its affinity at pH 7.4, is transcytosed through
blood-brain barrier endothelial cells, whereas another antibody showing
equally efficient binding at both pH values to the transferrin receptor,
is degraded inside the cell. The invention allows the selection and
generation of antibodies against transcytosis receptors that avoid
intracellular degradation in endothelial or epithelial barrier cells by a
modified sorting behavior caused by pH-dependent, reversible binding to
those receptors.

I. Definitions

[0047] The term "affinity" denotes the strength of the sum total of
non-covalent interactions between a single binding site of a molecule
(e.g. a polypeptide or an antibody) and its binding partner (e.g. a
target or an antigen). Unless indicated otherwise, as used herein,
"binding affinity" refers to intrinsic binding affinity which reflects a
1:1 interaction between members of a binding pair (e.g. in a
polypeptide-polynucleotide-complex, or between a polypeptide and its
target, or between an antibody and its antigen). The affinity of a
molecule X for its partner Y can generally be represented by the
dissociation constant (Kd). Affinity can be measured by common methods
known in the art, such as surface plasmon resonance and also including
those reported herein. A higher affinity of a molecule X for its binding
partner Y can be seen by a lower Kd and/or EC50 value.

[0048] The term "antibody" encompasses the various forms of antibody
structures including whole antibodies and antibody fragments. The
antibody as reported and used herein can be a human antibody, a humanized
antibody, a chimeric antibody, or a T cell antigen depleted antibody. The
term "antibody" refers to a protein consisting of one or more
polypeptide(s) substantially encoded by immunoglobulin genes. The
recognized immunoglobulin genes include the different constant region
genes as well as the myriad immunoglobulin variable region genes.
Immunoglobulins may exist in a variety of formats, including, for
example, Fv, Fab, and F(ab)2 as well as single chains (scFv) or
diabodies. A full length antibody in general comprises two so called
light chain polypeptides (light chain) and two so called heavy chain
polypeptides (heavy chain). Each of the heavy and light chain
polypeptides contains a variable domain (variable region) (generally the
amino terminal portion of the polypeptide chain) comprising binding
regions that are able to interact with an antigen. Each of the heavy and
light chain polypeptides comprises a constant region (generally the
carboxyl terminal portion). The constant region of the heavy chain
mediates the binding of the antibody i) to cells bearing a Fc gamma
receptor (FcγR), such as phagocytic cells, or ii) to cells bearing
the neonatal Fc receptor (FcRn) also known as Brambell receptor. It also
mediates the binding to some factors including factors of the classical
complement system such as component (Clq).

[0049] The variable domain of an immunoglobulin's light or heavy chain in
turn comprises different segments, i.e. four framework regions (FR) and
three hypervariable regions (CDR).

[0050] The term "binding pair" denotes a polypeptide, which comprises an
antibody heavy chain variable domain and an antibody light chain variable
domain. The variable domains can be connected to each other by any
suitable means such as a peptide bond, a linker, or a linking
non-peptidic component. In one embodiment the binding pair is selected
from Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, linear antibodies,
single-chain antibody molecules, and multispecific antibodies formed from
antibody fragments, full length heavy chain, full length light chain,
complete antibody, bispecific antibody, trispecific antibody,
tetraspecific antibody, or hexaspecific antibody. In one embodiment the
binding pair is a monoclonal antibody. In one embodiment binding pair is
at least a fragment of a complete antibody, a member of the
immunoglobulin superfamily, or a polypeptide with immunoglobulin-like
structure, that retains the binding specificity for its antigen.

[0051] The term "binding site" denotes a polypeptide that can specifically
bind to another polypeptide. In one embodiment the binding site is a
binding pair. In one embodiment the binding site is a polypeptide with
immunoglobulin-like modular structure, which can be selected from the
group consisting of fibronectin, TCR, CTLA-4, single-chain antigen
receptors, e.g. those related to T-cell receptors and antibodies,
antibody mimetics, transferrin, apolipoprotein, adnectins, molecules
based on anticalins, phylomers, avimers, affibodies, ankyrin repeats,
Kunitz domains, PDZ-domains, scorpio toxins immunity proteins, Knottins,
Versabodies, Green Fluorescent Protein and other non-antibody protein
scaffolds with antigen binding properties.

[0052] The term "CNS-related disease" denotes a disease or disorder of the
central nervous system (CNS). CNS-related diseases are, without being
limited to, particularly (i) neurodegenerative diseases or disorders such
as Parkinson's disease, Alzheimer's disease, or Huntington's disease,
(ii) psychiatric diseases such as depression, anxiety disorders,
schizophrenia, (iii) neuroinflammatory and other neurological disorders
such as multiple sclerosis, Amyotrophic Lateral Sclerosis, autism, or
pain; (iv) tumors of the CNS, or (v) viral and bacterial infections of
the CNS.

[0054] An "anti-angiogenic agent" refers to a compound which blocks, or
interferes with to some degree, the development of blood vessels. The
anti-angiogenic agent may, for instance, be a small molecule or an
antibody that binds to a growth factor or growth factor receptor involved
in promoting angiogenesis. The anti-angiogenic factor is in one
embodiment an antibody that binds to Vascular Endothelial Growth Factor
(VEGF).

[0056] The term "fMLP" denotes the tripeptide consisting of
N-formylmethionine, leucine and phenylalanine In one embodiment the
effector moiety is fMLP or a derivative thereof.

[0057] The term "fusion polypeptide" denotes a polypeptide that comprises
or is consisting of at least two discrete peptides or polypeptides that
are not found together in this way in a polypeptide in nature, i.e. these
portions are not occurring in the same polypeptide or in the same order
in nature. The portions of the fusion polypeptide are linked by a peptide
bond.

[0058] The term "peptidic linker" denotes linkers of natural and/or
synthetic origin comprising amino acid residues connected to each other
via peptide bonds. They consist of a linear amino acid chain wherein the
20 naturally occurring amino acids are the monomeric building blocks. The
chain has a length of from 1 to 50 amino acid residues, in one embodiment
between 3 and 28 amino acid residues, in a further embodiment between 4
and 20 amino acid residues. The linker may contain repetitive amino acid
sequences or sequences of naturally occurring polypeptides. The linker
has the function to ensure that the two components connected through the
linker can fold correctly and be presented properly due to steric and
rotational freedom. In one embodiment the linker is a "synthetic peptidic
linker" that is designated to be rich in glycine, glutamine, and/or
serine residues. These residues are arranged e.g. in small repetitive
units of up to five amino acids, such as (G)GGGS, (Q)QQQG, or (S)SSSG
(SEQ ID NO: 1, 2, and 3). This small repetitive unit may be repeated for
two to five times to form a multimeric unit. Other synthetic peptidic
linkers are composed of a single amino acid, that is repeated between 10
to 20 times, such as e.g. serine in the linker GSSSSSSSSSSSSSSSG (SEQ ID
NO: 4). In one embodiment the linker is selected from [GQ4]3GNN
(SEQ ID NO: 5), LSLSPGK (SEQ ID NO: 6), LSPNRGEC (SEQ ID NO: 7), LSLSGG
(SEQ ID NO: 8), LSLSPGG (SEQ ID NO: 9), G3[SG4]2SG (SEQ ID
NO: 10), or G3[SG4]2SG2 (SEQ ID NO: 11).

[0059] The term "prodrug" refers to a precursor or derivative form of a
pharmaceutically active substance that is less cytotoxic to tumor cells
compared to the parent drug and is capable of being enzymatically
activated or converted into the more active parent form. See, e.g.,
Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society
Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella,
et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery,"
Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana
Press (1985). The prodrugs that can be used as effector moiety include,
but are not limited to, phosphate-containing prodrugs,
thiophosphate-containing prodrugs, sulfate-containing prodrugs,
peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated
prodrugs, β-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
5-fluorouridine prodrugs which can be converted into the more active
cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized
into a prodrug form for use in this invention include, but are not
limited to, those chemotherapeutic agents described herein.

[0061] An "effective amount" of an agent, e.g., a pharmaceutical
formulation, refers to an amount effective, at dosages and for periods of
time necessary, to achieve the desired therapeutic or prophylactic
result.

[0062] The term "EC50-value" denotes the half maximal effective
concentration of a polypeptide, e.g. an antibody, that induces a response
of 50% between the baseline value and the maximum value in a
determination system, e.g. an ELISA. This is a measure of a therapeutic
drug's potency. Thus, the EC50-value is the concentration that is
calculated based on experimental data corresponding to the concentration
of a drug substance resulting in 50% effect. Decreasing EC50-values
denotes a higher affinity and potency of the drug.

[0063] A "human antibody" is an antibody which possesses an amino acid
sequence which corresponds to that of an antibody produced by a human or
a human cell or derived from a non-human source that utilizes human
antibody repertoires or other human antibody-encoding sequences. This
definition of a human antibody specifically excludes a humanized antibody
comprising non-human antigen-binding residues.

[0064] A "humanized" antibody refers to a chimeric antibody comprising
amino acid residues from non-human HVRs and amino acid residues from
human FRs. In certain embodiments, a humanized antibody will comprise
substantially all of at least one, and typically two, variable domains,
in which all or substantially all of the HVRs (e.g. CDRs) correspond to
those of a non-human antibody, and all or substantially all of the FRs
correspond to those of a human antibody. A humanized antibody optionally
may comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization.

[0065] An "immunoconjugate" is an antibody or antibody fragment conjugated
to one or more non antibody derived molecules, including but not limited
to a member of a binding pair, a nucleic acid, or an effector moiety.

[0066] An "individual" or "subject" is a mammal. Mammals include, but are
not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and
horses), primates (e.g., humans and non-human primates such as monkeys),
rabbits, and rodents (e.g., mice and rats). In certain embodiments, the
individual or subject is a human.

[0068] The term "monoclonal antibody" refers to an antibody obtained from
a population of substantially homogeneous antibodies, i.e., the
individual antibodies comprising the population are identical and/or bind
the same epitope, except for possible variant antibodies, e.g.,
containing naturally occurring mutations or arising during production of
a monoclonal antibody preparation, such variants generally being present
in minor amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. Thus, the modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of the
antibody by any particular method. For example, the monoclonal antibodies
or monoclonal antibody fragments to be used in the fusion polypeptide as
reported herein may be made by a variety of techniques, including but not
limited to the hybridoma method, recombinant DNA methods, phage-display
methods, and methods utilizing transgenic animals containing all or part
of the human immunoglobulin loci, such methods and other exemplary
methods for making monoclonal antibodies being described herein.

[0069] The term "pharmaceutical formulation" refers to a preparation which
is in such form as to permit the biological activity of an active
ingredient contained therein to be effective, and which contains no
additional components which are unacceptably toxic to a subject to which
the formulation would be administered.

[0070] A "pharmaceutically acceptable carrier" refers to an ingredient in
a pharmaceutical formulation, other than an active ingredient, which is
nontoxic to a subject. A pharmaceutically acceptable carrier includes,
but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0071] The term "transcellular transport" denotes a multistep process in
which a molecule, especially a macromolecule or biopolymer such as an
antibody, is transported across the cytosol of a cell. In the first step
of a transcellular transport material/molecules of the extracellular
space or cell surface-associated or -bound molecules are enclosed in a
vesicle. This step is called endocytosis. The vesicle diffuses across the
cytosol of the cell. Thereafter the endocytotic step is reversed, i.e.
the vesicle is fused with the cell membrane and the interior of the
vesicle is released to the extracellular space. Transcellular transport
takes place for example in epithelial cells, cells of the
blood-brain-barrier, neurons, or intestinal cells.

[0072] As used herein, "treatment" (and grammatical variations thereof
such as "treat" or "treating") refers to clinical intervention in an
attempt to alter the natural course of the individual being treated, and
can be performed either for prophylaxis or during the course of clinical
pathology. Desirable effects of treatment include, but are not limited
to, preventing occurrence or recurrence of disease, alleviation of
symptoms, diminishment of any direct or indirect pathological
consequences of the disease, preventing metastasis, decreasing the rate
of disease progression, amelioration or palliation of the disease state,
and remission or improved prognosis. In some embodiments, fusion
polypeptides as reported herein are used to delay development of a
disease or to slow the progression of a disease.

[0073] The term "variable region" or "variable domain" refers to the
domain of an antibody heavy or light chain that is involved in binding
the antibody to its antigen. The variable domains of the heavy chain and
light chain (VH and VL, respectively) of a native antibody generally have
similar structures, with each domain comprising four conserved framework
regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt,
T. J., et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y.
(2007), page 91). A single VH or VL domain may be sufficient to confer
antigen-binding specificity. Furthermore, antibodies that bind a
particular antigen may be isolated using a VH or VL domain from an
antibody that binds the antigen to screen a library of complementary VL
or VH domains, respectively (see, e.g., Portolano, S., et al., J.
Immunol. 150 (1993) 880-887, Clackson, T., et al., Nature 352 (1991)
624-628).

[0074] The term "vector," as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic acid
structure as well as the vector incorporated into the genome of a host
cell into which it has been introduced. Certain vectors are capable of
directing the expression of nucleic acids to which they are operatively
linked. Such vectors are referred to herein as "expression vectors."

[0075] The articles "a" and "an" are used herein to refer to one or to
more than one (i.e., to at least one) of the grammatical object of the
article. By way of example, "an antibody" means one antibody or more than
one antibody.

[0076] A "polypeptide" is a polymer consisting of amino acids joined by
peptide bonds, whether produced naturally or synthetically. Polypeptides
of less than about 20 amino acid residues may be referred to as
"peptides", whereas molecules consisting of two or more polypeptides or
comprising one polypeptide of more than 100 amino acid residues may be
referred to as "proteins". A polypeptide may also comprise non-amino acid
components, such as carbohydrate groups, metal ions, or carboxylic acid
esters. The non-amino acid components may be added by the cell, in which
the polypeptide is expressed, and may vary with the type of cell.
Polypeptides are defined herein in terms of their amino acid backbone
structure or the nucleic acid encoding the same. Additions such as
carbohydrate groups are generally not specified, but may be present
nonetheless.

[0077] The term "specifically binding" denotes that the binding site or
polypeptide or antibody or antibody fragments binds to its target with an
dissociation constant (Kd) of 10-5 M or less, in one embodiment of
from 10-7 M to 10-13 M, in a further embodiment of from
10-7 M to 10-9 M. The term is further used to indicate that the
polypeptide does not bind to other biomolecules present, i.e. it binds to
other biomolecules with a dissociation constant (Kd) of 10-4 M or
more, in one embodiment of from 10-4M to 1 M.

[0078] The term "pharmaceutically active compound" denotes any molecule or
combination of molecules whose activity it is desired to be delivered to
a place of action. Pharmaceutically active compounds include, but are not
limited to drugs (e.g. polypeptides, antibodies), labels, cytotoxins
(e.g. Pseudomonas exotoxin, ricin, abrin, Diphtheria toxin, and the
like), enzymes, growth factors, transcription factors, radionuclides,
ligands, liposomes, nanoparticles, viral particles, cytokines, and the
like.

II. Compositions and Methods

[0079] Herein is reported a fusion polypeptide with which it is possible
to transport therapeutics (biologically active compounds) such as
polypeptides, antibodies, or toxins, across a cell membrane, especially
the blood-brain-barrier. The fusion polypeptide as reported herein
employs therefore a general transport mechanism, i.e. receptor-mediated
endocytosis and transcytosis using an internalizing cell surface
receptor.

[0080] Herein is reported as one embodiment a fusion polypeptide
comprising [0081] at least one binding site, which binds to an
internalizing cell surface receptor, and [0082] at least one
pharmaceutically active compound, [0083] whereby the ratio of the
EC50-value of the binding site that binds to an internalizing cell
surface receptor determined at pH 5.5 and the EC50-value of the same
binding site to the same receptor determined at pH 7.4 is 10 or more.

[0084] In one embodiment the ratio is 15 or more.

[0085] In one embodiment the ratio is 100 or less.

[0086] In one embodiment the ratio is 10 to 100.

[0087] In one embodiment the binding site has an EC50-value
determined at pH 5.5 of 700 ng/ml or more. In one embodiment the binding
site has an EC50-value determined at pH 5.5 of 850 ng/ml or more. In
one embodiment the binding site has an EC50-value determined at pH
5.5 of 1000 ng/ml or more.

[0088] In one embodiment the binding site is a binding pair, which
comprises an antibody heavy chain variable domain and an antibody light
chain variable domain. In one embodiment the binding pair is selected
from Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, linear antibodies,
single-chain antibody molecules, and multispecific antibodies formed from
antibody fragments, full length heavy chain, full length light chain,
complete antibody, bispecific antibody, trispecific antibody,
tetraspecific antibody, or hexaspecific antibody. In one embodiment the
binding pair is a monoclonal antibody. In one embodiment the binding pair
is at least a fragment of a complete antibody, a member of the
immunoglobulin superfamily, or a polypeptide with immunoglobulin-like
structure, that retains the binding specificity for its antigen.

[0090] In one embodiment the binding site is a full length antibody or
antibody fragment that specifically binds to the transferrin receptor.

[0091] Without being bound by theory FIG. 1 shows a schematic drawing of
the pH-dependent transcytosis mechanism. Iron-loaded holo-transferrin
(middle panel) is endocytosed with the transferrin receptor from the
apical membrane of the brain endothelial cells. Upon endosomal
acidification, iron is released from the holo-transferrin, initiated by a
conformational change in transferrin-binding domain of the receptor.
Apo-transferrin remains bound to the receptor. After passing through the
sorting endosome, the receptor is either recycled to the apical membrane
or transcytosed to the basolateral membrane. After vesicle-membrane
fusion, apo-transferrin, which has no affinity for the receptor at pH
7.4, dissociates from the receptor and leaves the cell. In contrast (left
panel), the transferrin-receptor antibody mAb 128.1, which binds the
receptor with high affinity at pH 7.4 as well as at pH 5.5, i.e. has an
EC50 value ratio of less than 5 (1.3), forms a tight complex with
the receptor that is also stable at the pH value in the endosome. The
presence of the pH-stable complex prevents recycling and transcytosis,
but rather induces re-routing of the receptor into CD63-positive late
endosomes. Anti-transferrin-receptor antibodies with a pH-dependent
binding profile (exemplified by antibody MEM-189, which shows reduced
receptor binding at reduced (acid) pH values as at endosomal pH; right
panel, EC50 value ratio of more than 10 (15.6)) can undergo
transcytosis and recycling, without being bound by theory probably by
reversible, low affinity interaction with the transferrin-receptor in the
endosomal compartments.

[0092] In FIG. 3 the validation of the transcytosis assay as used herein
by transcytosis of 125I-transferrin through hCMEC/D3 brain
endothelial cells is shown. hCMEC/D3 cells on collagen-coated filter
inserts were loaded with 125I-labeled transferrin for one hour.
Afterwards the inserts were washed and transferred to a new plate at
37° C. (FIG. 3A) or 4° C. (FIG. 3B). At the indicated time
points, radioactivity in the cell lysates (black squares), the apical
(grey columns) and basolateral (white columns) medium compartments was
measured by gamma counting (CPM) after TCA precipitation. The sum of the
radioactivity values for each time point is shown in white triangles.
While at 37° C., transferrin is leaving the cell layer at equal
amounts into apical or basolateral medium compartments (A), it stays
inside the cells at 4° C. (B), demonstrating that the transport
process is energy-dependent.

[0093] In FIG. 4 it is shown that mAb 128.1 against the human transferrin
receptor does not leave hCMEC/D3 cells. 125I-labeled mAb 128.1 was
allowed to be taken up by hCMEC/D3 cells and radioactivity was determined
in cellular, apical and basolateral medium compartments as described
above (FIG. 3). No intact antibody leaves the cells into the apical or
basolateral compartments. Instead, intracellular radioactivity is slowly
decreasing, providing a hint to intracellular degradation of the
antibody.

[0094] In FIG. 5 it is shown that mAb 128.1 against the human transferrin
receptor, unlike transferrin, co-localizes with late endosomal marker
CD63 after internalization. hCMEC/D3 cells grown on collagen-coated
coverslips were incubated with mAb 128.1 or FITC-labeled transferrin for
ten minutes and then processed for immunofluorescence. mAb 128.1 was
detected with Alexa-488-labeled secondary antibody (A), panel C shows
transferrin-FITC fluorescence. Both preparations were counterstained with
an antibody against the late endosomal marker CD63 and an
Alexa-594-labeled secondary antibody (B, D). While mAb 128.1 shows a
co-localization with CD63, transferrin is not found in the
late-endosomal/lysosomal compartment, indicating re-routing of the
transferrin receptor away from a recycling/transcytosis to a degradative
trafficking pathway by mAb 128.1

[0095] In FIG. 6 it is shown that an antibody against human IGF-1 receptor
(anti-IGF-1R antibody) is not transcytosed, but recycled to the
extracellular medium. The transcytosis experiment was conducted as
described above (see FIGS. 2 and 3) with the exception that antibody
quantification was not done by radioactivity counting but by using a
highly sensitive human IgG ELISA. It can be seen that the anti-IGF-1R
antibody is not transcytosed, but recycled to the apical compartment,
demonstrating that IGF-1 receptor is exclusively recycled in blood-brain
barrier endothelial cells.

[0096] In FIG. 7 it is shown that mAb MEM-189 against the human
transferrin receptor is, unlike mAb 128.1, recycled and transcytosed. The
experiment was done as described above (see FIG. 6), using a mouse IgG
ELISA for quantification. In contrast to mAb 128.1, which was not found
in the apical or basolateral compartments (see above, FIG. 4), mAb
MEM-189 is recycled and transcytosed to equal amounts, with a transfer
rate slightly lower than that of transferrin (see also FIG. 3A).

[0097] In FIG. 8 it is shown that mAb MEM-189 binds in a pH-dependent
fashion to the transferrin receptor, whereas mAb 128.1 does not show a
pH-dependent binding. Binding of antibodies 128.1 and MEM-189 to the
human transferrin receptor extracellular domain at pH 7.4 (extracellular
pH) or pH 5.5 (endosomal pH) was measured by ELISA. While mAb 128.1 binds
to the receptor at both pH values with similar affinity (triangles pH
7.4, crosses pH 5.5), mAb MEM-189 shows a strongly diminished binding at
pH 5.5 (inverted triangles) as compared to pH 7.4 (circles). At pH 7.4,
binding of mAb MEM-189 to the receptor is weaker than that of mAb 128.1
(see EC50-values in the following Table). In the following table the
EC50 values for various antibodies against cell surface receptors
and their respective EC50 value ratios are shown.

[0098] In FIG. 9 it is shown that mAbs 128.1 and MEM-189 compete for the
same epitope on the transferrin receptor. The transferrin receptor
extracellular domain was coated to a microtiter plate and pre-incubated
with mAbs 128.1 or MEM-189, before the binding of the respective other
mAb was detected. Binding of mAb MEM-189 to the receptor is fully blocked
by mAb 128.1 pre-incubation (inverted triangles) as compared to binding
in the absence of mAb 128.1 (circles). In contrast, binding of mAb 128.1
to the receptor is not inhibited by pre-incubation with mAb MEM-189
(triangles and crosses, respectively). In conclusion, mAb MEM-189 and mAb
128.1 compete for the same epitope on the human transferrin receptor. The
fact that mAb MEM-189 cannot prevent binding of mAb 128.1 can be
explained by the significantly higher affinity of mAb 128.1.

[0099] In FIG. 10 it is shown that antibodies M-A712 and 13E4 against the
human transferrin receptor, both of which do not display pH-dependent
binding, are not transcytosed through hCMEC/D3 cells.

[0100] The data presented above clearly demonstrate that the essential
feature for antibody transcytosis is not the receptor epitope but the
binding affinity to the receptor and the pH-dependent variation of the
binding affinity.

1. Affinity

[0101] In certain embodiments, the binding site of the fusion polypeptide
provided herein has a dissociation constant (Kd) of ≦10 μM,
≦1 μM, ≦100 μM, ≦10 nM, or ≦1 nM (e.g.
in one embodiment of from about 10 -5 M to about 10-9 M, or in
another embodiment of about 10-7 M or less, e.g. from 10-7 M to
10-13 M, e.g., from 10-9 M to 10-13 M).

[0102] In one embodiment, Kd is measured by a radiolabeled antigen binding
assay (RIA) performed wherein the binding site is a Fab fragment of an
antibody and its antigen as described by the following assay. Solution
binding affinity of FABs for antigen is measured by equilibrating Fab
with a minimal concentration of (125I)-labeled antigen in the
presence of a titration series of unlabeled antigen, then capturing bound
antigen with an anti-Fab antibody-coated plate (see, e.g., Chen, Y., et
al., J. Mol. Biol. 293 (1999) 865-881). To establish conditions for the
assay, MICROTITER® multi-well plates (Thermo Scientific) are coated
overnight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs)
in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2%
(w/v) bovine serum albumin in PBS for two to five hours at room
temperature (approximately 23° C.). In a non-adsorbent plate (Nunc
#269620), 100 pM or 26 pM [125I]-antigen are mixed with serial
dilutions of a Fab of interest (e.g., consistent with assessment of the
anti-VEGF antibody, Fab-12, in Presta, L. G., et al., Cancer Res. 57
(1997) 4593-4599). The Fab of interest is then incubated overnight;
however, the incubation may continue for a longer period (e.g., about 65
hours) to ensure that equilibrium is reached. Thereafter, the mixtures
are transferred to the capture plate for incubation at room temperature
(e.g., for one hour). The solution is then removed and the plate washed
eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the
plates have dried, 150 μl/well of scintillant (MICROSCINT-20®;
Packard) is added, and the plates are counted on a TOPCOUNT® gamma
counter (Packard) for ten minutes. Concentrations of each Fab that give
less than or equal to 20% of maximal binding are chosen for use in
competitive binding assays.

[0103] According to another embodiment, Kd is measured using surface
plasmon resonance assays using a BIACORE®-2000 or a BIACORE
®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. with
immobilized antigen CM5 chips at ˜10 response units (RU). Briefly,
carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are
activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the
supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH
4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of
5 μl/minute to achieve approximately 10 response units (RU) of coupled
protein. Following the injection of antigen, 1 M ethanolamine is injected
to block unreacted groups. For kinetics measurements, two-fold serial
dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%
polysorbate 20 (TWEEN-20®) surfactant (PBST) at 25° C. at a
flow rate of approximately 25 μ/min. Association rates (kon) and
dissociation rates (koff) are calculated using a simple one-to-one
Langmuir binding model (BIACORE® Evaluation Software version 3.2) by
simultaneously fitting the association and dissociation sensorgrams. The
equilibrium dissociation constant (Kd) is calculated as the ratio
koff/kon. See, e.g., Chen, Y., et al., J. Mol. Biol. 293 (1999) 865-881).
If the on-rate exceeds 106 M-1 s-1 by the surface plasmon
resonance assay above, then the on-rate can be determined by using a
fluorescent quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16
nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab
form) in PBS, pH 7.2, in the presence of increasing concentrations of
antigen as measured in a spectrometer, such as a stop-flow equipped
spectrophotometer (Aviv Instruments) or a 8000-series SLM-AMINCO®
spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

[0104] In certain embodiments, a fusion polypeptide as reported herein
comprises an antibody fragment as binding site. Antibody fragments
include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv,
and scFv fragments, and other fragments described below. For a review of
certain antibody fragments, see Hudson, P. J., et al., Nat. Med. 9 (2003)
129-134. For a review of scFv fragments, see, e.g., Plueckthun, A., In:
The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore
(eds.), Springer-Verlag, New York (1994), pp. 269-315; see also WO
93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of
Fab and F(ab')2 fragments comprising salvage receptor binding epitope
residues and having increased in vivo half-life, see U.S. Pat. No.
5,869,046.

[0106] Single-domain antibodies are antibody fragments comprising all or a
portion of the heavy chain variable domain or all or a portion of the
light chain variable domain of an antibody. In certain embodiments, a
single-domain antibody is a human single-domain antibody (Domantis, Inc.,
Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

[0107] Antibody fragments can be made by various techniques, including but
not limited to proteolytic digestion of an intact antibody as well as
production by recombinant host cells (e.g. E. coli or phage), as
described herein.

3. Multispecific Antibodies

[0108] In certain embodiments, a fusion polypeptide as reported herein is
a multispecific fusion polypeptide, e.g. a bispecific fusion polypeptide.
Multispecific fusion polypeptides have binding specificities for at least
two different sites. In certain embodiments, one of the binding
specificities is for an internalizing cell surface receptor and the other
is for a therapeutic target. In certain embodiments, a bispecific fusion
polypeptide may bind to two different epitopes of the internalizing cell
surface receptor. Bispecific fusion polypeptides can be prepared as full
length fusion polypeptide or fusion polypeptide fragment.

[0109] In one embodiment the binding site of the fusion polypeptide is a
complete antibody.

[0111] Engineered antibodies with three or more functional antigen binding
sites, including "Octopus antibodies," are also included herein (see,
e.g. US 2006/0025576A1).

[0112] The antibody or fragment can be a "Dual Acting FAB" or "DAF"
comprising an antigen binding site that binds to the internalizing cell
surface receptor as well as another, different antigen (see, US
2008/0069820, for example).

[0114] In certain embodiments, a fusion polypeptide as reported herein may
be further modified to contain additional non-proteinaceous moieties that
are known in the art and readily available. The moieties suitable for
derivatization of the fusion polypeptide include but are not limited to
water soluble polymers. Non-limiting examples of water soluble polymers
include, but are not limited to, polyethylene glycol (PEG), copolymers of
ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,
polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,
poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids
(either homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propylene glycol homopolymers,
polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols
(e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene
glycol propionaldehyde may have advantages in manufacturing due to its
stability in water. The polymer may be of any molecular weight, and may
be branched or unbranched. The number of polymers attached to the
antibody may vary, and if more than one polymer is attached, they can be
the same or different molecules. In general, the number and/or type of
polymers used for derivatization can be determined based on
considerations including, but not limited to, the particular properties
or functions of the antibody to be improved, whether the antibody
derivative will be used in a therapy under defined conditions, etc.

[0115] In another embodiment, conjugates of a fusion polypeptide and
non-proteinaceous moiety that may be selectively heated by exposure to
radiation are provided. In one embodiment, the non-proteinaceous moiety
is a carbon nanotube (Kam, N. W., et al., Proc. Natl. Acad. Sci. USA 102
(2005) 11600-11605). The radiation may be of any wavelength, and
includes, but is not limited to, wavelengths that do not harm ordinary
cells, but which heat the non-proteinaceous moiety to a temperature at
which cells proximal to the antibody-non-proteinaceous moiety are killed.

5. Assays

[0116] Fusion polypeptides as reported herein or components thereof may be
identified, screened for, or characterized for their physical/chemical
properties and/or biological activities by various assays known in the
art.

5.1. Binding Assays and Other Assays

[0117] In one aspect, the binding site of the fusion polypeptide as
reported herein is tested for its cell surface receptor binding activity,
e.g., by known methods such as ELISA, Western blot, etc.

[0118] In another aspect, competition assays may be used to identify
further binding sites, especially antibodies and antibody fragments that
competes with mAb MEM-189 for binding to the transferrin receptor. In
certain embodiments, such a competing antibody binds to the same epitope
(e.g., a linear or a conformational epitope) that is bound by mAb
MEM-189. Detailed exemplary methods for mapping an epitope to which an
antibody binds are provided in Morris, G. E., (ed.), "Epitope Mapping
Protocols," In: Methods in Molecular Biology, Vol. 66, Humana Press,
Totowa, N.J. (1996).

[0119] An "antibody that binds to the same epitope" as a reference
antibody refers to an antibody that blocks binding of the reference
antibody to its antigen in a competition assay by 50% or more, and
conversely, the reference antibody blocks binding of the antibody to its
antigen in a competition assay by 50% or more.

[0120] For example, a fusion protein of the human transferrin receptor
extracellular domain linked to human IgGl Fc can be coated to a 96-well
plate by incubating 50 μl of a solution of 1 μg/ml in PBS for 1 h
at RT. After 1 h of blocking with PBS/1% (w/v) BSA and four washes with
PBS/0.1% (w/v) Tween, the antibody in question can be added to the plate
at different concentrations in PBS/0.1% (w/v) BSA adjusted to pH 7.4 or
pH 5.5 and incubated for 1.5 h at RT. After four washes with PBS/0.1%
(w/v) Tween, bound antibodies can be detected using HRP-coupled secondary
antibodies (30 min., RT) and 50 μl of TMB substrate. Color development
can be stopped by addition of 50 μl of 1 N hydrochloric acid (HCl) and
absorbance can be measured at 450 nm in a plate reader.

[0122] hCMEC/D3 cells (passages 26-29) can be cultured to confluence on
collagen-coated coverslips (microscopy) or flasks in EBM2 medium
containing 2.5% FBS, a quarter of the supplied growth factors and fully
complemented with supplied hydrocortisone, gentamycin and ascorbic acid.

[0123] For all transcytosis assays, high density pore
(1×108pores/cm2) PET membrane filter inserts (0.4 μm
pore size, 12 mm diameter) can be used in 12-well cell culture plates.
Media volumes are calculated to be 400 μl and 1600 μl for apical
and basolateral chambers, respectively. Apical chambers of filter inserts
can be coated with rat tail collagen I (7.5 μg/cm2) followed by
fibronectin (5 μg/ml), each incubation lasting for 1 h at RT. hCMEC/D3
cells can be grown to confluent monolayers (˜2×105
cells/cm2) for 10-12 days in EBM2 medium. Empty filters can be
blocked in PBS containing 1% BSA for 1 h or overnight (o/n) before assay
and then calibrated for at least 1 h in EBM2 before the assay.

[0124] The assay (see FIG. 2 for assay scheme) can be performed in
serum-free EBM2 medium which was otherwise reconstituted as described
herein. On day of the assay, cells are serum-starved for 60 min. to
deplete the natural ligand of the internalizing cell surface receptor in
question. Filter inserts with or without (but blocked overnight in
complete medium) cells were incubated apically with radiolabeled natural
ligand of the internalizing cell surface receptor, 125I-labeled or
unlabeled monoclonal antibodies in question for 1 h at 37° C.
Afterwards the entire apical and basolateral volume are collected.
Paracellular flux can be calculated from the determined values. The
monolayers were washed at RT in serum-free medium apically (400 μl)
and basolaterally (1600 μl) three times for 3-5 min. each. All wash
volumes were collected to monitor efficiency of removal of the unbound
ligand or antibody. Pre-warmed medium was added to the apical chamber and
the filters transferred to a fresh 12 well plate (blocked overnight with
PBS containing 1% BSA) containing 1600 μl pre-warmed medium. At this
point, filters with or without cells were lysed in 500 μl RIPA buffer
in order to determine specific ligand or antibody uptake. The remaining
filters were incubated at 37° C. or at 4° C. and samples
collected at various time points to determine apical and/or basolateral
release of ligand or antibody. Intact and degraded 125I-labeled
natural ligand or 125I-labeled antibody was assessed using trichloro
acetic acid (TCA) precipitation. The quantity of radioactive natural
ligand or antibody in supernatants or lysates can be determined by
gamma-radiation counting. The content of unlabeled antibody in the
samples can be quantified using a highly sensitive IgG ELISA (see Example
3). For each time point, data should be generated from two empty filters
and three filter cell cultures.

6. Recombinant Methods and Compositions

[0125] Fusion polypeptides may be produced using recombinant methods and
compositions. In one embodiment, isolated nucleic acid encoding a fusion
polypeptide as reported herein is provided. In a further embodiment, one
or more vectors (e.g., expression vectors) comprising such nucleic acid
are provided. In a further embodiment, a host cell comprising such
nucleic acid is provided. In one such embodiment, a host cell comprises
(e.g., has been transformed with) one or more vector comprising a nucleic
acid that encodes an amino acid sequence comprising the fusion
polypeptide. In one embodiment, the host cell is eukaryotic, e.g. a
Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, SP2/0
cell). In one embodiment, a method of making a fusion polypeptide as
reported herein is provided, wherein the method comprises culturing a
host cell comprising a nucleic acid encoding the fusion polypeptide, as
provided above, under conditions suitable for expression of the fusion
polypeptide, and optionally recovering the fusion polypeptide from the
host cell (or host cell culture medium).

[0126] For recombinant production of a fusion polypeptide as reported
herein, nucleic acid encoding a fusion polypeptide as reported herein,
e.g., as described above, is isolated and inserted into one or more
vectors for further cloning and/or expression in a host cell. Such
nucleic acid may be readily isolated and sequenced using conventional
procedures.

[0127] Suitable host cells for cloning or expression of
polypeptide-encoding vectors include prokaryotic or eukaryotic cells
described herein. For example, polypeptide may be produced in bacteria,
in particular when glycosylation is not needed. For expression of
antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.
Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, K. A., In:
Methods in Molecular Biology, Vol. 248, Lo, B. K. C., (ed.), Humana
Press, Totowa, N.J. (2003), pp. 245-254, describing expression of
antibody fragments in E. coli.) After expression, the polypeptide may be
isolated from the bacterial cell paste in a soluble fraction and can be
further purified.

[0128] In addition to prokaryotes, eukaryotic microbes such as filamentous
fungi or yeast are suitable cloning or expression hosts for
polypeptide-encoding vectors, including fungi and yeast strains whose
glycosylation pathways have been "humanized," resulting in the production
of a fusion polypeptide with a partially or fully human glycosylation
pattern. See Gerngross, T. U., Nat. Biotech. 22 (2004) 1409-1414, and Li,
H., et al., Nat. Biotech. 24 (2006) 210-215.

[0129] Suitable host cells for the expression of glycosylated polypeptides
are also derived from multicellular organisms (invertebrates and
vertebrates). Examples of invertebrate cells include plant and insect
cells. Numerous baculoviral strains have been identified which may be
used in conjunction with insect cells, particularly for transfection of
Spodoptera frugiperda cells.

[0132] Herein are also provided fusion polypeptides in which at least one
of the components such as the effector moiety is e.g. a cytotoxic agent,
such as a chemotherapeutic agent or drug, a growth inhibitory agent, a
toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial,
fungal, plant, or animal origin, or fragments thereof), or a radioactive
isotope.

[0135] In another embodiment the effector moiety is a radioactive atom. A
variety of radioactive isotopes are available for the production of
radioconjugates. Examples include At211, I131, I125,
Y90, Re186, Re188, Sm153, Bi212,
P32Pb212, and radioactive isotopes of Lu. When the
radioconjugate is used for detection, it may comprise a radioactive atom
for scintigraphic studies, for example Tc99m or I123, or a spin
label for nuclear magnetic resonance (NMR) imaging (also known as
magnetic resonance imaging, MRI) I123 again, such as I131,
In111, F19, C13, N15, O17, gadolinium, manganese
or iron.

[0136] The effector moiety can be fused to the binding site in the fusion
polypeptide as reported herein using a variety of bifunctional protein
coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate
(SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate
(SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido components (such
as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such
as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine components (such as
1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be
prepared as described in Vitetta, E. S., et al., Science 238 (1987)
1098-1104. Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene
triamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent for
conjugation of radionucleotide to the fusion polypeptide (see WO
94/11026). The linker for conjugating the toxic moiety to the fusion
polypeptide as reported herein can be a "cleavable linker" facilitating
release of a cytotoxic drug in the cell. For example, an acid-labile
linker, peptidase-sensitive linker, photolabile linker, dimethyl linker,
or disulfide-containing linker (Chari, R. V., et al., Cancer Res. 52
(1992) 127-131, U.S. Pat. No. 5,208,020) can be used.

[0137] The effector moiety can be fused to the binding site in the fusion
polypeptide as reported herein via a linker, which is e.g. but not
limited to such conjugates prepared with cross-linker reagents including,
but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP,
SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS,
sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB
(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available
(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA).

[0138] The effector moiety can be fused via a peptidic linker to the
binding site. In one embodiment the peptidic linker has from 4 to 20
amino acid residues. In one embodiment the linker is the same between the
repeat-motif-molecules, in another embodiment the conjugate contains
linker with two or more different amino acid sequences. In a further
embodiment the linker is selected from (G3S), (G3S)2,
(G3S)3, (G3S)4, (G3S)5, (G4S),
(G4S)2, (G4S)3, (G4S)4, (G4S)5
(SEQ ID NO: 1 and SEQ ID NO: 12 to 20), especially from (G4S)3
and (G4S)4 (SEQ ID NO: 18 and SEQ ID NO: 19).

8. Methods and Compositions for Diagnostics and Detection

[0139] In certain embodiments any of the fusion polypeptides provided
herein is useful for detecting the presence of a target specifically
bound by a binding site in the fusion polypeptide in a biological sample.
The term "detecting" as used herein encompasses quantitative or
qualitative detection.

[0140] In one embodiment the fusion polypeptide is provided for use in a
method of diagnosis or detection. In a further aspect, a method of
detecting the presence of the target of the binding site or the effector
moiety of the fusion polypeptide as reported herein in a biological
sample is provided. In certain embodiments, the method comprises
contacting the biological sample with a fusion polypeptide as reported
herein under conditions permissive for binding of the binding site(s) or
the effector moiety to its target(s), and detecting whether a complex is
formed between the fusion polypeptide and the target. Such method may be
an in vitro or in vivo method. In one embodiment the fusion polypeptide
as reported herein is used to select subjects eligible for therapy with
an isolated polypeptide comprised in the fusion polypeptide, e.g. where
the target is a biomarker for selection of patients.

[0141] In certain embodiments a labeled fusion polypeptide is provided,
i.e. a fusion polypeptide wherein the effector moiety is a label. Labels
include, but are not limited to, labels that are detected directly (such
as fluorescent, chromophoric, electron-dense, chemiluminescent, and
radioactive labels), as well as labels, such as enzymes or ligands, that
are detected indirectly, e.g., through an enzymatic reaction or molecular
interaction. Exemplary labels include, but are not limited to, the
radioisotopes P32, C14, I125, H3, and I131,
fluorophores such as rare earth chelates or fluorescein and its
derivatives, rhodamine and its derivatives, dansyl, umbelliferone,
luciferases, e.g., firefly luciferase and bacterial luciferase (U.S. Pat.
No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish
peroxidase (HRP), alkaline phosphatase, β-galactosidase,
glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,
galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic
oxidases such as uricase and xanthine oxidase, coupled with an enzyme
that employs hydrogen peroxide to oxidize a dye precursor such as HRP,
lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,
bacteriophage labels, stable free radicals, and the like.

III. Pharmaceutical Formulations

[0142] Pharmaceutical formulations of a fusion polypeptide as reported
herein are prepared by mixing such fusion polypeptide having the desired
degree of purity with one or more optional pharmaceutically acceptable
carriers (Osol, A., (ed.) Remington's Pharmaceutical Sciences 16th
edition (1980)), in the form of lyophilized formulations or aqueous
solutions. Pharmaceutically acceptable carriers are generally nontoxic to
recipients at the dosages and concentrations employed, and include, but
are not limited to: buffers such as phosphate, citrate, and other organic
acids; antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyl dimethylbenzyl ammonium chloride, hexamethonium
chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or
benzyl alcohol, alkyl parabens such as methyl or propyl paraben,
catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low
molecular weight (less than about 10 residues) polypeptides, proteins,
such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers
such as polyvinyl pyrrolidone, amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine, monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose, or
dextrins, chelating agents such as EDTA, sugars such as sucrose,
mannitol, trehalose or sorbitol, salt-forming counter-ions such as
sodium, metal complexes (e.g. Zn-protein complexes), and/or non-ionic
surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically
acceptable carriers herein further include interstitial drug dispersion
agents such as soluble neutral-active hyaluronidase glycoproteins
(sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins,
such as rhuPH2O (HYLENEX®, Baxter International, Inc.). Certain
exemplary sHASEGPs and methods of use, including rhuPH2O, are described
in US 2005/0260186 and US 2006/0104968. In one aspect, a sHASEGP is
combined with one or more additional glycosaminoglycanases such as
chondroitinases.

[0143] Exemplary lyophilized antibody formulations are described in U.S.
Pat. No. 6,267,958. Aqueous antibody formulations include those described
in U.S. Pat. No. 6,171,586 and WO 2006/044908, the latter formulations
including a histidine-acetate buffer.

[0144] The formulation herein may also contain more than one active
ingredients as necessary for the particular indication being treated,
especially those with complementary activities that do not adversely
affect each other. Such active ingredients are suitably present in
combination in amounts that are effective for the purpose intended.

[0146] Sustained-release preparations may be prepared. Suitable examples
of sustained-release preparations include semi-permeable matrices of
solid hydrophobic polymers containing the antibody, which matrices are in
the form of shaped articles, e.g. films, or microcapsules.

[0147] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.

IV. Therapeutic Methods and Compositions

[0148] Any of the fusion polypeptides as reported herein wherein the
effector moiety is a therapeutically active compound or a detectable
label may be used in therapeutic methods.

[0149] In one aspect a fusion polypeptide as reported herein for use as a
medicament is provided. In further aspects a fusion polypeptide for use
in treating CNS-related disease is provided. In certain embodiments a
fusion polypeptide for use in a method of treatment is provided. In
certain embodiments the invention provides a fusion polypeptide for use
in a method of treating an individual having a CNS-related disease
comprising administering to the individual an effective amount of the
fusion polypeptide. In one such embodiment the method further comprises
administering to the individual an effective amount of at least one
additional therapeutic agent. In further embodiments the invention
provides a fusion polypeptide as reported herein for use in reversing or
stabilizing of a CNS-related disease. In certain embodiments, the
invention provides a fusion polypeptide for use in reversing or
stabilizing of a CNS-related disease in an individual comprising
administering to the individual an effective amount of the fusion
polypeptide to reverse or stabilize a CNS-related disease. An
"individual" according to any of the above embodiments is especially a
human.

[0154] In one embodiment the effector moiety is a therapeutically active
compound selected from an anticonvulsant, an anxiolytic agent, a
cytokine, or a polynucleotide such as siRNA.

[0155] In a further aspect as reported herein is provided the use of a
fusion polypeptide as reported herein in the manufacture or preparation
of a medicament. In one embodiment the medicament is for treatment of a
CNS-related disease. In a further embodiment the medicament is for use in
a method of treating a CNS-related disease comprising administering to an
individual having a CNS-related disease an effective amount of the
medicament. In a further embodiment the medicament is for reversing or
stabilizing a CNS-related disease. In a further embodiment the medicament
is for use in a method of reversing or stabilizing a CNS-related disease
in an individual comprising administering to the individual an amount of
the medicament effective to reverse or stabilize a CNS-related disease.
An "individual" according to any of the above embodiments may be a human.

[0156] In a further aspect as reported herein a method for treating a
CNS-related disease is provided. In one embodiment the method comprises
administering to an individual having such a disease an effective amount
of a fusion polypeptide as reported herein. An "individual" according to
any of the above embodiments may be a human.

[0157] In a further aspect as reported herein a method for reversing or
stabilizing a CNS-related disease in an individual is provided. In one
embodiment the method comprises administering to the individual an
effective amount of a fusion polypeptide as reported herein to reverse or
stabilize a CNS-related disease. In one embodiment an "individual" is a
human.

[0158] In a further aspect as reported herein a pharmaceutical formulation
comprising any one of the fusion polypeptides provided herein, e.g., for
use in any of the above therapeutic methods is provided. In one
embodiment the pharmaceutical formulation comprises any one of the fusion
polypeptides provided herein and a pharmaceutically acceptable carrier.
In another embodiment the pharmaceutical formulation comprises any one of
the fusion polypeptides as reported herein and at least one additional
therapeutic agent.

[0159] Fusion polypeptides as reported herein can be used either alone or
in combination with other agents in a therapy. For instance, a fusion
polypeptide as reported herein may be co-administered with at least one
additional therapeutic agent.

[0160] Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included in the
same or separate formulations), and separate administration, in which
case, administration of the antibody of the invention can occur prior to,
simultaneously, and/or following, administration of the additional
therapeutic agent and/or adjuvant. Fusion polypeptides as reported herein
can also be used in combination with radiation therapy.

[0161] A fusion polypeptide as reported herein can be administered by any
suitable means, including parenteral, intrapulmonary, and intranasal,
and, if desired for local treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous, intraarterial,
intraperitoneal, or subcutaneous administration. Dosing can be by any
suitable route, e.g. by injections, such as intravenous or subcutaneous
injections, depending in part on whether the administration is brief or
chronic. Various dosing schedules including but not limited to single or
multiple administrations over various time-points, bolus administration,
and pulse infusion are contemplated herein.

[0162] Fusion polypeptides as reported herein would be formulated, dosed,
and administered in a fashion consistent with good medical practice.
Factors for consideration in this context include the particular disorder
being treated, the particular mammal being treated, the clinical
condition of the individual patient, the cause of the disorder, the site
of delivery of the agent, the method of administration, the scheduling of
administration, and other factors known to medical practitioners. The
fusion polypeptide need not be, but is optionally formulated with one or
more agents currently used to prevent or treat the disorder in question.
The effective amount of such other agents depends on the amount of fusion
polypeptide present in the formulation, the type of disorder or
treatment, and other factors discussed above. These are generally used in
the same dosages and with administration routes as described herein, or
about from 1% to 99% of the dosages described herein, or in any dosage
and by any route that is empirically/clinically determined to be
appropriate.

[0163] For the prevention or treatment of disease, the appropriate dosage
of a fusion polypeptide as reported herein (when used alone or in
combination with one or more other additional therapeutic agents) will
depend on the type of disease to be treated, the type of fusion
polypeptide, the severity and course of the disease, whether the fusion
polypeptide is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to the
fusion polypeptide, and the discretion of the attending physician. The
fusion polypeptide is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of the
disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of fusion
polypeptide can be an initial candidate dosage for administration to the
patient, whether, for example, by one or more separate administrations,
or by continuous infusion. One typical daily dosage might range from
about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned
above. For repeated administrations over several days or longer,
depending on the condition, the treatment would generally be sustained
until a desired suppression of disease symptoms occurs. One exemplary
dosage of the fusion polypeptide would be in the range from about 0.05
mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0
mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be
administered to the patient. Such doses may be administered
intermittently, e.g. every week or every three weeks (e.g. such that the
patient receives from about two to about twenty, or e.g. about six doses
of the fusion polypeptide). An initial higher loading dose, followed by
one or more lower doses may be administered. The progress of this therapy
can be easily monitored by conventional techniques and assays.

Articles of Manufacture

[0164] In another aspect as reported herein an article of manufacture
containing materials useful for the treatment, prevention and/or
diagnosis of the disorders described above is provided. The article of
manufacture comprises a container and a label or package insert on or
associated with the container. Suitable containers include, for example,
bottles, vials, syringes, IV solution bags, etc. The containers may be
formed from a variety of materials such as glass or plastic. The
container holds a composition which is by itself or combined with another
composition effective for treating, preventing and/or diagnosing the
condition and may have a sterile access port (for example the container
may be an intravenous solution bag or a vial having a stopper pierceable
by a hypodermic injection needle). At least one active agent in the
composition is a fusion polypeptide as reported herein. The label or
package insert indicates that the composition is used for treating the
condition of choice. Moreover, the article of manufacture may comprise
(a) a first container with a composition contained therein, wherein the
composition comprises a fusion polypeptide as reported herein; and (b) a
second container with a composition contained therein, wherein the
composition comprises a further cytotoxic or otherwise therapeutic agent.
The article of manufacture in this embodiment of the invention may
further comprise a package insert indicating that the compositions can be
used to treat a particular condition. Alternatively, or additionally, the
article of manufacture may further comprise a second (or third) container
comprising a pharmaceutically-acceptable buffer, such as bacteriostatic
water for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials desirable
from a commercial and user standpoint, including other buffers, diluents,
filters, needles, and syringes.

[0165] The following examples, sequence listing and figures are provided
to aid the understanding of the present invention, the true scope of
which is set forth in the appended claims. It is understood that
modifications can be made in the procedures set forth without departing
from the spirit of the invention.

[0184] For all transcytosis assays, high density pore
(1×108pores/cm2) PET membrane filter inserts (0.4 μm
pore size, 12 mm diameter) were used in 12-well cell culture plates.
Media volumes were calculated to be 400 μl and 1600 μl for apical
and basolateral chambers, respectively. Apical chambers of filter inserts
were coated with rat tail collagen I (7.5 μcm2) followed by
fibronectin (5 μg/ml), each incubation lasting for 1 h at RT. hCMEC/D3
cells were grown to confluent monolayers (˜2×105
cells/cm2) for 10-12 days in EBM2 medium. Empty filters were blocked
in PBS containing 1% BSA for 1 h or overnight (o/n) before assay and then
calibrated for at least 1 h in EBM2 medium before the assay.

Example 2

Transcytosis Assay of 125I-transferrin and Monoclonal Antibodies

[0185]125I-transferrin (Tfn) was obtained from Perkin Elmer (Perkin
Elmer, Rodgau, Germany, #NEX212050UC). mAb 128.1 against the human and
mAb 8D3 against the mouse transferrin receptor were transiently expressed
in HEK cells transfected with a vector comprising a continuous open
reading frame of the coding sequences of human IgG1 heavy and light chain
constant regions, respectively, and the variable regions of the mouse
anti-human transferrin-receptor antibody 128.1 (for variable region
sequences see WO 93/10819 and SEQ ID NO: 21 and 22) or the rat anti-mouse
transferrin-receptor antibody 8D3 (Boado et al. (2009), Biotechnol.
Bioeng. 102, 1251-1258) and purified as reported previously. mAb 128.1
was also labeled with 125I. A monoclonal antibody against the human
IGF-1 receptor was expressed and purified as described in U.S. Pat. No.
7,572,897. Mouse monoclonal mAbs MEM-189 and 13E4 against the human
transferrin receptor were obtained from Abcam (Cambridge, England,
#ab1086 and #ab38171, respectively) and mAb M-A712 from BD Biosciences
(Heidelberg, Germany, #555534). The entire assay (see FIG. 2 for assay
scheme) was performed in serum-free EBM2 medium which was otherwise
reconstituted as described in Example 1. On day of the assay, cells were
serum-starved for 60 min. to deplete transferrin (only for transferrin
transcytosis). Filter inserts with or without (but blocked overnight in
complete medium) cells were incubated apically with radiolabeled
transferrin, 125I-labeled or unlabeled monoclonal antibodies for 1 h
at 37° C. Afterwards the entire apical and basolateral volume were
collected. Paracellular flux and stability of the radio-iodinated ligand
were calculated from the determined values. The monolayers were washed at
RT in serum-free medium apically (400 μl) and basolaterally (1600
μl) three times for 3-5 min. each. All wash volumes were collected to
monitor efficiency of removal of the unbound ligand or antibody.
Pre-warmed medium was added to the apical chamber and the filters
transferred to a fresh 12 well plate (blocked overnight with PBS
containing 1% BSA) containing 1600 μl pre-warmed medium. At this
point, filters with or without cells were lysed in 500 μl RIPA buffer
(Sigma, Munich, Germany, #R0278) in order to determine specific ligand or
antibody uptake. The remaining filters were incubated at 37° C. or
at 4° C. and samples collected at various time points to determine
apical and/or basolateral release of ligand or antibody. Intact and
degraded 125I-transferrin or 125I-mAb 128.1 was assessed using
trichloro acetic acid (TCA) precipitation. The quantity of radioactive
transferrin or mAb 128.1 in supernatants or lysates was determined by
gamma-radiation counting. The content of unlabeled antibody in the
samples was quantified using a highly sensitive IgG ELISA (see Example
3). For each time point, data was generated from two empty filters and
three filter cell cultures.

Example 3

Sensitive IgG ELISA after Transcytosis Assay

[0186] The entire procedure was performed at RT using an automated washer
for the wash steps. A 384-well plate was coated with 30 μl/well of 1
μg/ml anti-human/mouse-IgG, Fcγ-specific (Dianova, Hamburg,
Germany, #109-005-098 or #115-005-164, respectively) in phosphate
buffered saline solution (PBS) for 2 h followed by 1 h incubation in
blocking buffer PBS containing 1% (w/v) BSA (Sigma, Munich, Germany,
#A2153) for human and mouse IgG assays, respectively. Serially diluted
samples from the transcytosis assay and standard concentrations of the
antibody used in the transcytosis assay were added to the plate and
incubated for 2 h. After four washes, 30 μl/well of 50 ng/ml
anti-human/mouse-F(ab)2-biotin-conjugate (Dianova, Hamburg, Germany,
#109-066-097 or #115-066-072, respectively) in blocking buffer (see
above) was added and incubated for a further 2 h. Following six washes,
30 μl/well of 50 ng/ml (huIgG assay) or 100 ng/ml (mIgG assay)
poly-HRP40-streptavidin (Fitzgerald, Acton (MA), USA, #65R-S104PHRPx; in
PBS containing 1% (w/v) BSA and 0.05% (w/v) Tween-20) was added and
incubated for 30 min. After four washes, immune complexes were detected
by addition of 30 μl/well of BM Chemiluminescence Substrate (Roche
Diagnostics GmbH, Mannheim, Germany). The luminescence signal was
measured using a luminescence plate reader and concentration calculated
using the fitted standard curve. The range of the assay was from 10 pg/ml
to 10 ng/ml.

Example 4

Confocal Fluorescence Microscopy

[0187] To investigate the localization of the 128.1 antibody and
holo-transferrin, monolayers of hCMEC/D3 cells grown to confluence on
collagen-coated coverslips were incubated with 5 μg/ml FITC-tagged
holo-transferrin (Invitrogen, Darmstadt, Germany, #T-2871) or 1 μg/ml
mAb 128.1 for 10 min. Thereafter the medium was removed and replaced with
fresh medium. After 1 h at 37° C., the monolayers were fixed in 4%
paraformaldehyde (PFA) for 15 min. at RT, permeabilized for 10 min.
(PBS/0.1% (w/v) Triton X-100 (Sigma, Munich, Germany, #93443) and
incubated with an antibody against late endosomal/lysosomal marker CD63
(R&D Systems, Wiesbaden, Germany, #MAB5417) for 45 min. at RT. Cells were
subjected to washes in PBS/0.1% (w/v) Triton X-100 for 15 min. and
sequentially incubated, where necessary, with secondary antibodies (goat
anti-human IgG-Alexa Fluor 488 and/or chicken anti-mouse IgG-Alexa Fluor
594 (Invitrogen, Darmstadt, Germany, #A11013 or #A21201, respectively)
for 45 min. at RT. Cells were washed in PBS/0.1% (w/v) Triton X-100 for
30 min. Afterwards cover slips were mounted in mounting medium.
Fluorescent images were obtained using a confocal microscope. All the
confocal images show a single, representative, section of a Z-series
taken through the entire cell.

Example 5

pH-dependent Binding and Competition ELISA

[0188] A fusion protein of the human transferrin receptor extracellular
domain linked to human IgG1 Fc (R&D Systems, Wiesbaden, Germany, #
2474-TR-050) or the extracellular domain of the mouse transferrin
receptor (SinoBiological, Beijing, China, #50741-MO7H) or of the human
insulin receptor (R&D Systems, Wiesbaden, Germany, #1544-IR/CF) were
coated to a 96-well plate by incubating 50 μl of a solution of 1
μg/ml in PBS for 1 h at RT. After 1 h of blocking with PBS/1% (w/v)
BSA and four washes with PBS/0.1% (w/v) TWEEN 20, antibodies MEM-189,
128.1, 13E4, M-A712, LT-71, MEM-75 (both Abcam, #ab9179 and #ab38446,
respectively), OKT-9 (eBioscience, Frankfurt, Germany, #16-0719; all
against the human TfR), 8D3, R17217 (Santa Cruz, Heidelberg, Germany,
#sc-52504; both against the mouse TfR), 83-13 (Invitrogen, Darmstadt,
Germany, #AHR0221) and 243524 (R&D Systems, #MAB1544; both against the
human insulin receptor) were added to the plate at different
concentrations in PBS/0.1% (w/v) BSA adjusted to pH 7.4 or pH 5.5 and
incubated for 1.5 h at RT. Alternatively, wells were incubated with mAb
MEM-189 or mAb 128.1 as blocking antibodies at a fixed concentration of 5
μg/ml, washed four times, and afterwards incubated for 30 min. at RT
with different concentrations of the other antibody that had not been
used for blocking After four further washes with PBS/0.1% (w/v) TWEEN 20,
bound antibodies were detected using HRP-coupled secondary antibodies
(Dianova, Hamburg, Germany, #109-036-097 or GE Healthcare, Freiburg,
Germany, #NA9310V)(30 min., RT) and 50 μl of TMB (10 min., RT)
substrate. Color development was stopped by addition of 50 μl of 1 N
hydrochloric acid (HCl) and absorbance measured at 450 nm in a plate
reader.